Methods of treating hyperinsulinism



United States Patent 3,475,534 METHODS OF TREATING HYPERINSULINISMRichard L. Fenichel, Wyncote, and Harvey E. Alburn,

West Chester, Pa., assignors to American Home Products Corporation, NewYork, N.Y., a corporation of Delaware No Drawing. Filed Jan. 23, 1967,Ser. No. 610,739 Int. Cl. A61k 17/02, /00

US. Cl. 424-177 1 Claim ABSTRACT OF THE DISCLOSURE Summary 0 theinvention.-This invention relates to compositions for, and means totreat, hyperinsulinism. More particularly, it is concerned with theadministration of a complex of reduced insulin B chains with albumin towarm-blooded animals suffering from hyperinsulinism to securesymptomatic relief in cases of insulin overdosages or in situationswherein the body supplies too much insulin for the subjects normalneeds. The instant compositions are also useful in screening andselecting thereapeutic substances effective in the treatment ofdiabetes, and as diagnostic aids in determining whether diabetic orprediabetic conditions exist in individuals.

Background of the invenzioru.Hyperinsulinism occurs under manycircumstances. Pancreatic islet cell adenoma, pancreatic islet cellcarcinoma and diffuse hyperphasia of islet cells are the most dramaticconditions that produce frank hyperinsulinism. These insulin secretingtumors are met in all age groups and even in veterinary medicine. Mildhyperinsulinism, moreover, occurs in individuals with hypertension. Inaddition, stress and emotional disturbances produce a symptom complexresulting in fatigue in which hyperinsulinism is an outlet mechanism.Increased intestinal hormone secretion of secretin and pancreazymin alsoaugment insulin release.

In hyperinsulinism, the blood sugar falls and the symptoms are theresult of hypoglycemia. Thus patients suifering from hyperinsulinism canexhibit the following symptoms: sweating, pallor, flushing, salivation,tachycardia, anxiety, confusion, irritability, negativism, dullness,excitement, violence, stammering and other speech disorders diplopia,nystagmus, anisocoria, tremor, transient paralysis, positive Babinskisign, aphasia, incoordination, twitching, convulsions, epigastricsinking sensation, hunger, increase in hunger contractions in bloodsugar levels down to 75 mgm. percent, gastric atony below that level,occasional vomiting, slight rise in blood pressure, anginal pain inpatients with damaged myocardium, and the like. The hypoglycemiasyndrome, if untreated, terminates in coma and death. Thus, it is clearthat the availability of a specific anti-insulin agent would be of greatvalue to directly and immediately combat hyperinsulinism.

There is also a well known and clearly felt need to provide chemicalagents useful to treat diabetes, especially those which are effectiveorally, to replace insulin. Insulin suffers from shortcomings includingthe need to isolate it from animal organs, the need to keep it cold sothat loss in potency does not occur, the need to administer it by thepainful injection route, and the like. The selection of chemical agentsuseful to treat diabetes would be facilitated by a rapid, inexpensiveand convenient screening method and the instant compositions lendthemselves to use in such screens.

3,475,534 Patented Oct. 28, 1969 ice There also exists a need to providemeans to diagnose diabetes and prediabetes in suspected individuals.This would provide an additional aid to diagnosticians who now rely onmeasurements of blood sugar or urine sugar. It is a matter of commonknowledge and experience that individuals often are hyperglycemicwithout being diabetic. Thus among the more common non-diabetic causesof sugar in the blood or urine there are cases of true renal glycosuria(which is harmless and hereditary), renal glycosuria associated with amore extensive tubular dysfunction, and even temporary alimentary orstarvation glycosuria. Furthermore, there is now recognized in certainindividuals a condition known as pre-diabetes. Here, certain changes areoccurring in various body tissues despite normal diagnostic tests. Thesewill later develop into the classic diabetic degenerative vascularprocesses, but the condition goes unrecognized, usually, becauseprediabetes occurs in advance of detectable blood sugar abnormalities.Thus, a means to detect diabetes and to distinguish its symptoms fromnon-diabetic glycosurias and to detect pre-diabetes, would clearly bewelcome.

It is, accordingly, a principal object of this invention to provide ameans to relieve hyperinsulinism which is particularly effective,non-toxic and characterized by high levels of activity for extendedperiods of time. A further object is to provide novel hyperglycemicdosage forms with anti-insulin activity which are particularlyelfective, non-toxic and characterized by high levels of activity forextended periods of time. Still another primary object of the instantinvention is to provide novel and efficient means to screen and selecttherapeutic substances with anti-diabetic activity. Yet anotherprincipal object of this invention is to provide means to assist in thediagnosis of diabetes and prediabetes in suspected individuals.

Description of the invention-These objects are realized by use of acomposition which is, in essence: a hyperglycemic composition withanti-insulin activity comprising a complex of reduced insulin B chainpolypeptide with albumin and a pharmaceutical carrier, said compositionbeing in injectable dosage unit form.

The therapeutic means of this invention, broadly stated, is a method forthe treatment of a warm-blooded animal suifering from hyperinsulinismwhich comprises administering to said animal a composition ascontemplated hereinabove, the quantity of the said complex administeredbeing suflicient to alleviate the hyperinsulinism.

The screening means contemplated by this invention is, in essence, amethod for screening and selecting therapeutic substances effective totreat diabetes, which comprises introducing said substance into a systemincluding insulin and a complex of reduced insulin B chain With albuminand determining whether said substance blocks the inhibition, in vitroor in vivo, of insulin by said complex, those substances blocking saidinhibition being selected.

The diagnostic means contemplated by this invention is, in essence, amethod for diagnosing diabetic or predia'betic conditions inwarm-blooded animals which comprises administering a compositioncomprising a complex of reduced insulin B chain polypeptide with albuminand a pharmaceutical carrier to the suspected diabetic or prediabeticanimal and measuring the blood sugar rise, reporting as positive thosesuspects in which the blood sugar rise is substantially greater thanthat induced by administering said complex to non-diabetic ornon-prediabetic control animals.

The medicament complex employed in the compositions and methods of thisinvention comprises a reduced insulin B chain polypeptide associatedwith albumin. As is well known to those skilled in the art, insulin is aprotein, each molecule of which is a tetramer. Each of the fourmonomers, in turn, comprises two polypeptide chains linked to each otherby disulfide bridges between cysteine residues in adjacent chains. Thesepolypeptides are of two types, named A and B, respectively, one of eachbeing present in each monomer of the hormone. Not only is the amino acidcomposition of these peptide chains known, but the sequence in whichthey occur has been determined. Furthermore, it is easily possible toseparate the A and B chains by known methods and procedures for thiswill be fully described hereinafter. The term albumin contemplates achemical compound, protein in nature. These yield only amino acids uponcleavage with enzymes or acids. Illustrative albumins useful in thisinvention are grain alburmin, ovalbumin, soy bean albumin, serumalbumin, lactabumin, and the like. Especially useful because of its highactivity and purity is crystalline bovine albumin, readily availablefrom numerous commercial sources. The term reduced when used inassociation with insulin B chains herein and in the appended chainscontemplates a product obtained by treating the B chains, preferably intheir S-sulfo form, with a reducing agent, preferably and conveniently,a combination of cysteine and [tris (hydroxymethyl) aminomethanehydrochloride] hereinafter called tris in accordance with usage wellestablished in the art. As will be fully described hereinafter, thecomplex of reduced insulin B chain polypeptide with albumin is preparedby mixing solutions of the reduced insulin B chain polypeptide withappropriate quantities of albumin, preferably at a temperature of about70 F., and preferably to make a concentration of about 1 mg. of albuminper 1 mg. of reduced B chain. Appropriate quantities of albumin includeat least about 0.1 part of albumin per 1 part of reduced B chain as alower limit. With respect to an upper limit; this is not critical; alarge relative amount of albumin can be used, for example, 100 parts perpart by weight of reduced B chain will function, but there is noadvantage in using this much.

The instant compositions can be administered in a variety of injectabledosage forms. As with insulin, the route of administration determinesthe duration and rapidity of action. Patients are given injectionsaseptically by the subcutaneous route, but in coma intravenous orintramuscular injections would be employed to insure rapid action. Inlarge (70 kg.) animals, a convenient site of subcutaneous administrationis the thigh.

The daily dose requirements vary with the particular composition beingemployed, the severity of the symp toms being presented, and thewarm-blooded animal being treated. The dosage also varies with the sizeof the patient. The ordinarily effective dose is from about 0.01 mg./kg.to about 1.0 mg./kg. per day, preferably about 0.14 mg./kg. Of course,as in the case with insulin, each patient requires individual study bythe physician to determine the most efficacious time, number, and amountof individual daily doses. As with insulin, blood sugar and urine sugarestimations provide a guide for therapy with the instant compositions,the therapeutic objective in the case of the instant anti-insulincompositions being to raise the blood sugar level to normal levels andthen to maintain it.

For unit dosages the instant complexes are compounded into a variety oflargely aqueous injectable dosage forms containing various electrolytes,buffers, stabilizers and the like. Thus, for example, the aqueoussuspension of the complex can contain sodium chloride, sodium acetate,methyl para-hydroxybenzoate, glycerin, dibasic sodium phosphate, smallstabilizing amounts of phenol, metacresol and the like. An especiallyuseful dosage unit comprises sterile water as the carrier, eachmilliliter of which contains 1 mg. of reduced B chain complexed with 1mg. of albumin, 1 mg. of cysteine and 3 mg. of tris.

For use in screening and selecting therapeutic substances withantidiabetic activity, the instant complexes are formulated and embodiedin in vitro and in vivo systerns by standard techniques, illustrationsof which will be fully described hereinafter.

For use as diagnostic aids, the instant complexes are injected into thesuspected animals at dose levels in the range specified hereinabove. Theelevation in blood sugar is measured by techniques familiar to thoseskilled in the art (for example, the procedure of Hoffman, J. Biol.Chem, 120, 51, 1937, can be used). Diabetics and prediabetics respond toa dose of the complex with a substantially greater rise in blood sugarthan do controls. For example, it is not unusual to find approximatelydouble the blood sugar rise in diabetic and prediabetic animals afteradministration of the instant complex than that seen afteradministration of the complex to normal control animals.

Preferred embodiments of the invention.The following examples areintended to describe two means to obtain the instant medicamentcompositions in forms fully satisfactory for use in the methods of thisinvention.

Example 1 The S-sulfo form of the A and B insulin chains are prepared byreacting crystalline bovine insulin With a sodium sulphite-cupricsulphate-urea reagent according to the procedure of Dixon and Wardlaw(Nature, 188: 721- 724, 1960). The urea that precipitates with the A andB chains of insulin upon addition of ethanol is removed from thepreparation by dialysis before lyophilization of the insulin chains. Achains are separated from B chains by solubilization of the chainmixture in pH 7.4, 0.2 M tris [tris(hydroxymethyl) aminomethanehydrochloride] buffer, mechanical dialysis of this solution againstdistilled water, and subsequent adsorption on cross-linked dextran ofthe solution to remove the major portionof the insulin S-sulfo A chains.The solution containing the insulin S- sulfo B chains is thenlyophilized.

The insulin S-sulfo B chains are initially solubilized in pH 3.5 ureaformate buffer and are then reduced in 1 mg./ 1 ml. aliquots in pH 8.6,0.02 M tris=0.0062 M cysteine buffer by heating for 2 minutes in a nearboiling hot Water bath. The mitochondrical swelling activity of 5X10 Mbovine crystalline insulin (0.9 mg./5 ml.) is markedly inhibited by thisreduced insulin B chain complexed with an equal weight of bovinecrystalline albumin (1 mg. of each in 5 ml. test system).

Example 2 S-sulfo A and B chains of crystalline insulin are preparedaccording to the procedure of Dixon and Wardlaw (Nature, 188: 722,1960). After a cross linked polystyrene cation exchange resin columntreatment and separation of the A and B chains has been accomplished,removal of urea is effected by dialysis of the solution with agitationfor 3 hours against 4 changes (13 1.) each of cool (18 C.) distilledwater. The solutions are then shell frozen and lyophilized.

S-sulfo B chains, prepared as above are reduced in 0.0062 Mcysteine=0.02 M tris solutions at a concentration of l mg./ml. ofsolution, at pH 8.6, by heating the 1 m1. aliquots in a near boilingwater bath for 2 minutes. Immediately after the reduced B chains havecooled to 70 F., crystalline bovine albumin is added so thatconcentration of 1 mg. per ml. of albumin is present for each mg. ofreduced B chain.

The reduced B chain-albumin complex is then dialyzed against pH 8.6,0.125 M KC1=0.02 M tris solution at a 40:1 gradient for 1% hours.

One milligram of dialyzed reduced insulin B chain complexed with 1 mg.of crystalline bovine albumin in 5 ml. of 0.125 M KCl=0.02 M trissolution at pH 7.3, effectively inhibits the mitochondrial swelling of5X10 M bovine crystalline insulin (0.9 mg. in 5 ml. test system).

In the preliminary evaluation of the hyperglycemic compositions andmethods of this invention, and as a proof of their valuable activity,the in vivo effect of reduced insulin B chain complexed with bovinecrystalline albumin is determined in 200 g. normal male Sprague Dawleyrats maintained on a standard balanced diet, but fasted 18 hours priorto the experiment. Reduced B chain complexed with an equal weight ofbovine crystalline albumin in 0.5 ml. of 0.0124 M cysteine=0.02 M trisreducing solution (2 mg. of each per ml.) is injected intraperitoneally.A control solution consisting of the reducing solution containing thesame concentration of bovine crystalline albumin as the experimentalreduced B chain solution is administered to a control group of rats. Acontrol blood sample (0.1 ml.) and samples minutes, 30 minutes, 90minutes, 150 minutes and 210 minutes after injection are obtained byexternal heart puncture under light ether anaesthesia. An experimentalgroup of rats receiving the reduced B chain-albumin complex by theintravenous route is also included in this experiment. Blood samples areanalyzed for glucose content in the procedure of Hoffman (J. Biol.Chem., 120: 51, 1937).

Blood glucose elevation above the control values is observed 15 minutesafter intraperitoneal or intravenous administration of the reduced Bchain albumin complex. A further blood glucose increase above thecontrol values is observed for both experimental groups 30 minutes afterinjection. A decline in blood sugar is observed 90 minutes after theinjection of reduced B chain-albumin for both experimental groups, butthese blood glucose levels are still greater than the control values.The increase in blood sugar values for both experimental groups abovethe control blood sugar values persists throughout the experiment.

A second type of in vivo experiment is utilized using normal maleSprague Dawley rats maintained on a standard, balanced diet in order toobtain a sustained increase in blood sugar with administration ofreduced B chain complexed with albumin.

After an 18 hour fast, the animals are injected intraperitoneally with 1ml. of 0.0062 M cysteine=0.02 M tris solution at pH -8.6=8.9 containingeither 1 mg. of reduced B chain plus 1 mg. of albumin, or albumin alone.A second injection of either the same amount of reduced B chain andalbumin, or albumin alone is given after the 30 minute blood sample isobtained. Blood samples (0.1 ml.) are taken from the heart under lightether anaesthesia before the first injection and 30, 60 and 90 minutesthereafter. Blood glucose levels are determined as above. Under theseconditions the peak elevation of blood glucose of the reduced Bchain-albumin injected rats over that of the controls persists for 1hour as indicated by the 30 and 60 minute blood glucose values, beforefalling off at 90 minutes.

Methods for preparation of systems to screen and select, in vivo and invitro, therapeutic substances for antidiabetic activity are described inthe following examples:

Example 3 Rat liver mitochondria are prepared in 0.25 M sucrose solutioncontaining 0.001 M ethylenediamine tetraacetic acid-disodiumethylenediamineteraacetate (Judah, Biochim. Biophys. Acta, 53: 375,1961) using the differential centrifugation procedure of Schneider (J.Biol. Chem., 176: 259, 1948). The effect of 5X10 M concentration ofinsulin in the presence of reduced and dialyzed B chain-albumin complex(0.9 mg. of each) and a selected concentration of a test compound onmitochondrial swelling, in which the mitochondria are suspended in 0.125M KCl-0.02 M tris=0.l% partially hydrolyzed gelatin solution at pH 7.3(solution A), is measured by following changes in light absorption at520 ml with a Beckman Model B Spectrophotometer (Lehninger, A. L., J.Biol. Chem., 234: 2187, 1959). The insulin, reduced and dialyzed Bchain-albumin complex and selected concentrations of compound aresolubilized in 5 ml. of Solution A.

Controls are run using insulin, reduced B-chain-albumin plus compound,compound alone, and insulin plus re- Male Sprague Dawley rats weighing-200 g. are fed ad libitum for one week on a high fat, high protein dietof the following composition:

Casein, high nitrogen 315 Vegetable oil, hydrogenated 620 Salt U.S.P.XIV 62 B vitamins 1 Choline chloride 2 After one week the rats weighbetween and 200 g. The effect of reduced insulin B chain-albumin complexon blood glucose levels is determined after the rats have been fasted 18hours prior to the experiment. After obtaining'an initial 0.1 ml. bloodsample from the heart by external heart puncture under light etheranaesthesia the rats are given 0.5 ml. of a solution or a suspension ofa compound under test by intraperitoneal injection. The compound issolubilized in physiological saline, or a suitable buffer or suspended,for example, in carboxymethyl cellulose. After 10 minutes the animalsreceive 1 ml. of a 1 mg./-ml. solution of reduced insulin B chainalbumincomplex in 0.02 M tris--0.0062 M cysteine reducing solution pH 8.6 (5mg./kg.). A second intraperitoneal injection of reduced B chain-albumin(5 mg./ kg.) is given to these animals after obtaining a 30 minute bloodsample. Additional 0.1 ml. blood samples are obtained 60 and 90 minuteslater. Control animals treated in the same way as the experimentalanimals receive 1 ml. of 0.0062 M cysteine0.02 M this(hydroxymethyl)aminomethane hydrochloride solution containing 1 mg./ml.of crystalline bovine albumin instead of the reduced B chain-albumincomplex. Blood glucose values were determined by the procedure ofHoffman, cited hereinabove.

Additional control groups of rats receiving the reduced B chain-albumincomplex in reducing solution, or albumin alone in reducing solution, and0.5 ml. of physiological saline in the place of compound under test arealso included. The activity of a compound is determined by the extentthat it is able to prevent or alleviate the reduced B chain-albumininduced hyperglycemia. Those compounds which block the inhibition ofinsulin by the reduced B chain-albumin complex are reported as positive.

Example 5 To diagnose diabetes and prediabetes the in vivo testprocedure of Example 2 (second type) is repeated with maleSprague-Dawley rats. Those with diabetes and with prediabetes, and thosemaintained for 7 days on the high fat-high protein diet of Example 4(which mimics the conditions of starvation diabetes) elicit about twicethe blood glucose elevation observed in the normal diet, non-diabeticand non-prediabetic control animals at 30 and 60 minutes afteradministration of the complex and show a slower fall of glucoseelevation 90 minutes after administration of the complex.

We claim:

1. A method for the treatment of a warm-blooded animal suffering fromhyperinsulism which comprises administering to said animal by injectiona composition comprising (a) complex of a reduced S-sulfo form of theinsulin B chain polypeptide with from about 0.1 part 7 8 to about 100parts by weight of an albumin selected References Cited from the groupconsisting of grain albumin, ovalbumin, Lowy et all: The Lancet,8024304, 1961' soy bean albumin, serum albumin and lactalburnin perWilson et 31.: Biochemica et Biophysica Acta, 62 pp.

part by weight of said polypeptide and (b) a pharmaceuti- 483489 (1962)cal carrier, the amount of ingredient (a) in said composition beingsufficient to impart hyperglycemia activity 5 thereto, the quantity ofthe composition administered being sufficient to secure symptomaticrelief from the US, Cl, X R hyperinsulinism. 424 .178

FRANK CACCLXPAGLIA, JR., Primary Examiner

